While we currently know much about DNA genomic sequences as well as the functional significance of both coding and non-coding sequences, we have little ability to modulate these functions with sequence-specific, cell-permeable synthetic compounds. This deficiency is a barrier to applications of our genomic knowledge in biotechnology and therapeutic applications. The project described in this proposal will remove that barrier and will have an impact on human health through the potential for new anticancer and antiparasitic drugs as well as new applications in biotechnology. The project starts with an extensive library and knowledge of the DNA complexes of AT specific, minor groove binding compounds. We hypothesize that it is possible to use modules from the AT library and rationally couple novel modules for GC recognition to design compounds for broad, mixed sequence recognition of selected DNA target sequences. The AT specific compounds that we start with are cell permeable and are designed based on a molecular platform that includes clinically useful compounds. Our target compounds maintain these features while incorporating new GC base pair modules that are being designed in Aim 1 of the proposal. The remainder of Aim 1 describes preparation of entirely new types of modular compounds that use our known AT binding units with new GC recognition modules to bind tightly and specifically to a broad array of mixed sequences in DNA. As part of this Aim, we present some very promising preliminary results with several new types of DNA minor groove binders that can specifically recognize one or two GC base pairs in a mixed DNA sequence. These preliminary findings are a proof of concept that our modular design approach will work and that the approach can be expanded to more complex sequences as this project develops. Aim 2 of the project describes our methods for analysis of the DNA complexes of the new agents. We will start with a thermal melting screen that will separate strong-binding, specific-compounds from those that do not bind well to target DNA sequences. The second part of the Aim includes more detailed studies on the most important compounds from the screen. We will evaluate the interaction affinity, stoichiometry, kinetics and cooperativity of the better binding agents with biosensor-surface plasmon resonance, fluorescence spectroscopic, DNase I footprinting, mass spectrometry and calorimetric methods. The third part of Aim 2 includes structure determination by high resolution NMR methods and crystallographic determination of complex structures where crystals can be obtained. We will conduct limited exploratory studies of the ability of the compounds to inhibit important DNA-transition factor complexes and this is an important long term goal with very significant relevance for new drug development. The primary impact of this project will be the successful design of a library of motifs that can be linked in different combinations to recognize a broad array of biologically important mixed AT and GC bp DNA sequences in cells for new genomic applications.